Motor coordination refers to the ability to coordinate and integrate the movements of various parts of the body during movement. It integrates multiple physical qualities, including agility, speed, and balance, and fully reflects the central nervous system's control and regulation of muscle activity. In addition to central nervous system lesions, pathological changes in metabolism can affect an individual's motor coordination function. Therefore, motor coordination tests can often reflect pathological changes in the body.

In March 2024, Dr. Zhang Ran, currently at Tsinghua University, published a research paper titled "Regulation of the urea cycle by reversible high-stoichiometry lysine succinylation" in the internationally renowned journal Nature Metabolism (IF: 16.2) as the first author. Corresponding authors were Professor Eric Verdin of the University of California, San Francisco, and Professor Birgit Schilling of the Buck Institute for Research on Aging. The experiments, designed to verify animal motor coordination, utilized the rotarod, one of Ugo Basile's flagship products.

Background

Lysine succinylation is a post-translational modification of proteins. Succinylated proteins are widely involved in various metabolic pathways, including the tricarboxylic acid (TCA) cycle, the urea cycle, fatty acid oxidation, and ketone body synthesis. Therefore, succinylation plays a variety of potentially important roles in metabolic regulation and is associated with the pathogenesis of various diseases.

Currently, researchers have been able to identify thousands of succinylation sites within cells using antibody enrichment combined with proteomics analysis, but determining whether they are biologically significant is difficult. This study explored a method for globally identifying succinylation levels within cells, allowing rapid identification of functionally important, high-modification sites.

Experimental methods

Improved succinylation mass spectrometry sample preparation: Using GluC digestion, we found that after antibody enrichment, we could identify a number of modification sites comparable to trypsin, and GluC digestion identified a large number of novel succinylation modification sites (673).
       
Quantitative modification rate: SIRT5 (succinylation demodification enzyme) WT/KO mouse liver was lysed, and then the protein was fully introduced with heavy-labeled d4-succinic anhydride. After GluC enzymatic digestion, SWATH-DIA was used to detect the modification rate. Finally, the ratio of light-labeled to heavy-labeled succinylated modified peptides was used to determine the modification rate.


Phenotypic motor coordination function assessment: Testing was performed using the Ugo Basile rotarod apparatus on days 1, 14, and 28. Mice were acclimated for 30 minutes before the experiment. Each test consisted of a training phase and an experimental phase. During the training phase, mice remained on the rotarod at a constant speed of 5 rpm for 5 minutes. Mice were placed in separate lanes before the rotarod began to rotate (up to five mice could be tested simultaneously in a single experiment). The experiment was conducted three times, with a 30-minute interval between each session. The rotarod rotated slowly for the first 5 seconds of each experimental phase, followed by a gradual increase in speed from 5 to 50 rpm over the next 6 minutes. The experiment ended automatically if the mouse fell off the rod and was detected by the system; the experiment was manually recorded as ending when the mouse grasped the rod and rotated with it.

Experimental results

01. Identification of succinylation modification sites

822 succinylation modification sites were identified, and 238 sites that were also identified in the antibody enrichment results were further screened out. After analyzing the modification rates of these modification sites, it was found that the modification rates of most sites were between 0.1% and 5%, and 41 sites showed a modification rate of >5%.

02. Changes in modification levels after SIRT5 knockout

The succinylation modification levels at K112 and K121 of ASS1 protein increased most significantly after SIRT5 knockout, increasing from 2.3% and 18.1% to 39.9% and 39.6%, respectively.

03. Protein structure analysis

Only K121 is located around the active pocket of the ASS1 protein. Sequence analysis shows that K112 is not conserved among species, and only the K121E mutant protein will cause a significant decrease in the thermal stability and enzyme activity of ASS1, while the K112E mutation has basically no effect.

04. Urea cycle metabolism level detection

The levels of urea cycle-related metabolites in the livers of mice with SIRT5 knockout were significantly reduced, indicating that intracellular ASS1 activity was indeed reduced after SIRT5 knockout. The authors also found that SIRT5 knockout led to a decrease in the mice's ability to detoxify ammonia, while overexpressing ASS1 in the mouse liver could reduce the increase in ammonia levels caused by SIRT5 knockdown.

Experimental Conclusion

Using heavy succinic anhydride labeling combined with SWATH-DIA, the succinylation modification rate in rat liver was identified, and the highly modified protein ASS1 was identified. The Ugo Basile rotarod apparatus provided a powerful tool for phenotypic motor coordination function identification and provided important support for the experimental conclusions.

Ugo Basile Rotarod - Classic and easy to use, the gold standard for testing

The rotarod technique, originating from research published by NW Dunham and TSMiya in 1957, has proven valuable in screening for drugs with potential effects on motor coordination and function in mice and rats. The Ugo Basile Rotarod, the world's first rotarod developed based on this method for mice and rats, was named the "Rota-Rod." It is widely used in neuroscience research on Alzheimer's disease, ALS, Huntington's disease, brain damage, anxiety, and depression, and can also be applied to other studies investigating motor coordination.

01. Diverse models to meet different needs
Five rotation modes allow users to customize experimental protocols and adapt to a variety of animal models. Choose from: constant speed mode, linear acceleration mode, acceleration-deceleration mode, forward and reverse mode, and multi-stage rotation mode (this mode is user-defined).

02. Multi-channel operation, supporting simultaneous experiments on multiple animals

The mouse rotarod offers five channels, while the rat rotarod offers four channels. As mice in each channel fall from the rotating rod to the drop sensor below, the device automatically records rotation speed and endurance time. At the end of a multi-animal test, the display shows each animal's movement time, rotation mode, and rotation speed at the time of fall, as well as user-preset information.

03. Composite wheel, more sensitive testing
Developed jointly by Ugo Basile and Professor Bellesi of the University of Camerino in Italy, the optional installation of a composite wheel on the basic rotarod transforms the original uniform, continuous climbing surface into a stepped pattern of horizontal rods. The number of these rods can be increased, either symmetrically or asymmetrically, to vary the complexity of the test. According to recent publications, the composite wheel improves the sensitivity of the test, enabling the detection of even the smallest changes in motor deficits, making it particularly suitable for rats.

The RotaRod, a composite wheel for mice and rats, introduces additional complexity and improves sensitivity to motor coordination tests in mice and rats. Animals walk on a wheel with irregular horizontal bars, and the difficulty of the task can be controlled by randomly removing some bars, thereby adjusting the degree of motor cortex and hippocampal involvement. Available models for both mice and rats provide optimal experimental results.

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04. Wide range of applications and high number of literature citations
It can be applied to fatigue experiments, skeletal muscle relaxation experiments, central nervous system inhibition experiments, and other experiments that require exercise to detect drug effects, such as the effect of toxicity on exercise capacity, the effect of substance deficiency on exercise capacity, the effect of cardiovascular and cerebrovascular drugs on exercise capacity, etc.

It is often used in conjunction with other behavioral tests, such as grip strength and tail suspension tests, to measure an animal's muscle function, coordination, and balance. It has been cited in over 6,000 peer-reviewed articles and is considered the gold standard test for motor coordination research.

Related product recommendations

01.UGO Climbing Tester
The Ugo Basile Climbing Tester, jointly developed by Ugo Basile of Italy and Professor Sidney Negus's team at Virginia Commonwealth University, is an experimental instrument that automatically measures and quantifies vertical climbing behavior in mice. The Climbing Tester is designed to provide researchers with a simple, automated tool for investigating the vertical movement of small rodents within a cylinder.

02. UGO Grip Dynamometer

The Ugo Basile Grip Strength Meter is a classic device for assessing the grip strength of the forelimbs, hindlimbs, and all four limbs of mice and rats. It can be used to test the effects of muscle relaxants, neuroleptics, and stimulants on limb strength. It can also be used to assess animal aging, nerve damage, skeletal muscle damage, and postoperative recovery. It has a wide range of applications.

03.UGO fully automatic animal treadmill

The Ugo Basile fully automatic animal treadmill can be used for exercise fatigue testing in mice and rats across various disease models. Based on a belt-based runway, the device allows for forced exercise training and accurate fatigue testing of mice and rats by adjusting the belt speed and track inclination. All parameter settings are conveniently controlled via the instrument's touchscreen or accompanying software.

Some user documents

1.Achilly NP, Wang W, Zoghbi HY. Presymptomatic training mitigates functional deficits in a mouse model of Rett syndrome. Nature. 2021;592(7855):596-600. doi:10.1038/s41586-021-03369-7

2. Lieb A, Qiu Y, Dixon CL, et al. Biochemical autoregulatory gene therapy for focal epilepsy. Nat Med. 2018;24(9):1324-1329. doi:10.1038/s41591-018-0103-x

3.Cocozza G, di Castro MA, Carbonari L, et al. Ca2+-activated K+ channels modulate microglia affecting motor neuron survival in hSOD1G93A mice. Brain Behav Immun. 2018;73:584-595. doi:10.1016/j.bbi.2018.07.002

4.Gautam M, Jara JH, Kocak N, et al. Mitochondria, ER, and nuclear membrane defects reveal early mechanisms for upper motor neuron vulnerability with respect to TDP-43 pathology. Acta Neuropathol. 2019;137(1):47-69. doi:10.1007/s00401-018-1934-8

5. Weston M, Kaserer T, Wu A, et al. Olanzapine: A potent agonist at the hM4D(Gi) DREADD amenable to clinical translation of chemogenetics. Sci Adv. 2019;5(4):eaaw1567. Published 2019 Apr 17. doi:10.1126/sciadv.aaw1567

6. Elorza A, Márquez Y, Cabrera JR, et al. Huntington's disease-specific mis-splicing unveils key effector genes and altered splicing factors. Brain. 2021;144(7):2009-2023. doi:10.1093/brain/awab087

7. Alonso Bellido IM, Posada-Pérez M, Hernández-Rasco F, et al. Microglial Caspase-3 is essential for modulating hippocampal neurogenesis. Brain Behav Immun. 2023;112:206-219. doi:10.1016/j.bbi.2023.06.013

8. Alonso Bellido IM, Posada-Pérez M, Hernández-Rasco F, et al. Microglial Caspase-3 is essential for modulating hippocampal neurogenesis. Brain Behav Immun. 2023;112:206-219. doi:10.1016/j.bbi.2023.06.013

9.Haigh JL, Adhikari A, Copping NA, et al. Deletion of a non-canonical regulatory sequence causes loss of Scn1a expression and epileptic phenotypes in mice. Genome Med. 2021;13(1):69. Published 2021 Apr 26. doi:10.1186/s13073-021-00884-0

10. Zhang Y, Mickle AD, Gutruf P, et al. Battery-free, fully implantable optofluidic cuff system for wireless optogenetic and pharmacological neuromodulation of peripheral nerves. Sci Adv. 2019;5(7):eaaw5296. Published 2019 Jul 5. doi:10.1126/sciadv.aaw5296

Further Reading

Ugo Basile Climbing Tester | A New Vertical Motor Function Test: An Emerging Perspective on Pain Assessment in Rodents!

Ugo Basile | Thermal Pain Testing Experiment – Introduction to the Plantar Hot Spot Tester Experiment

A guide to the 50% withdrawal threshold for mice and rats: Accurate assessment of pain quantification

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